It is commonplace to grow animal cells in cell culture. Mass culture of animal cell lines is fundamental to the manufacture of vaccines and many biotechnology products. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, synthetic hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified) currently must be made in animal cells.
Many different cell types may be grown in cell culture including for example, stem cells, CHO cells and hybridoma cells. Stem cells differ from other kinds of cells in the body. Regardless of their source, all stem cells are capable of dividing and renewing themselves for long periods, are unspecialized, and can give rise to specialized cell types.
The specific factors and conditions that allow stem cells to remain unspecialized are of great interest. It has taken scientists many years of trial and error to derive and maintain stem cells in the laboratory without them spontaneously differentiating into specific cell types. It is thus desirable to elucidate the signals in a mature organism that cause a stem cell population to proliferate and remain unspecialized until the cells are needed. Such information is critical to be able to grow large numbers of unspecialized stem cells in the laboratory for further experimentation.
Stem cells are unspecialized in that stem cells do not have any tissue-specific structures that allow them to perform specialized functions; for example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and cannot carry oxygen molecules through the bloodstream (like a red blood cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.
Scientists are attempting to find new ways to control stem cell differentiation, thereby growing cells or tissues that can be used for specific purposes such as cell-based therapies or drug screening.
Adult stem cells typically generate the cell types of the tissue in which they reside; for example, a blood-forming adult stem cell in the bone marrow normally gives rise to the many types of blood cells. It is generally accepted that a blood-forming cell in the bone marrow (a hematopoietic stem cell) cannot give rise to the cells of a very different tissue, such as nerve cells in the brain. Experiments over the last several years have purported to show that stem cells from one tissue may give rise to cell types of a completely different tissue.
There are many ways in which human stem cells can be used in research and the clinic. Studies of human embryonic stem cells will yield information about the complex events that occur during human development. A primary goal is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs. Scientists know that turning genes on and off is central to this process. Some of the most serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation. A more complete understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. Predictably controlling cell proliferation and differentiation requires additional basic research on the molecular and genetic signals that regulate cell division and specialization. While recent developments with iPS cells suggest some of the specific factors that may be involved, techniques must be devised to introduce these factors safely into the cells and control the processes that are induced by these factors.
Human stem cells could also be used to test new drugs; for example, new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Other kinds of cell lines are already used in this way. Cancer cell lines, for example, are used to screen potential anti-tumor drugs. The availability of pluripotent stem cells would allow drug testing in a wider range of cell types. However, to screen drugs effectively, the conditions must be identical when comparing different drugs. Therefore, scientists will have to be able to control precisely the differentiation of stem cells into the specific cell type on which drugs will be tested. Current knowledge of the signals controlling differentiation falls short of being able to mimic these conditions precisely to generate pure populations of differentiated cells for each drug being tested.
Perhaps the most important potential application of human stem cells is the generation of cells and tissues that could be used for cell-based therapies. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.
The tremendous potential of hematopoietic stem/progenitor cells (HSPC) for reconstituting the hematopoietic system led to the development of HSPC transplantation as a clinical strategy for the treatment of hematological disorders and cancers. Bone marrow, mobilized peripheral blood, and umbilical cord blood are used as sources of transplantable HSPC (Bensinger et al., 2009; Gluckman. 2009). In an autologous transplantation, HSPC are collected from a patient to rescue the same patient from the effects of high dose-chemotherapy. In an allogeneic transplantation, HSPC are obtained from a donor (Bordignon et al., 2006). The low number of HSPC obtained from patients unable to mobilize sufficient number of HSPC to peripheral blood or a donor or a single cord blood unit limits their application. Therefore, many investigators have explored methods to expand HSPC ex vivo and various culture conditions, which included different cytokines and growth factors, have been proposed. There is general agreement on the key roles played by Flt-3 ligand, thrombopoietin, and stem cell factor in the regulation of the early stages of hematopoiesis (Conneally et al., 1997; Kaushansky et al., 1998; Shah et al., 1996; Petzer et al., 1996). However, attempts to expand HSPC ex vivo using hematopoietic growth factors have not achieved clinically relevant results (Dahlberg et al., 2011). More effective strategies for the amplification of cord blood or peripheral blood HSPC are needed to improve the therapeutic potential of clinical transplantation of HSPC and other cellular therapies.
There is a need in the art for methods to enhance proliferation of stem cells in cell culture to permit more rapid and efficient study of stem cells and their potential uses.
Animal cell cultures may also be used to produce monoclonal antibodies (mAbs) which may be used for treating cancers, Alzheimer's, auto-immune diseases, and various infectious diseases (Pandey, 2010; Rodrigues et al., 2010). This market is growing by 20.9% per year and reached $16.7 billion prior to 2008 (Birch et al., 2006). In 2010, global revenues from mAb-based products moved significantly past the $40 billion mark, and are poised to continue growing steadily. mAbs can be used for therapy by specifically binding to target cells, then stimulating the patient's immune system to attack diseased cells. mAb therapy can be used to destroy malignant tumor cells and prevent tumor growth by blocking specific cell receptors. Most monoclonal antibodies are produced by recombinant DNA technology or hybridomas.
Recombinant mAb production in mammalian cells is a complex multistep process requiring immunization of an animal, selection of specific B-lymphocytes, creation of cell lines (hybridoma or CHO cells), selection of a specific cell column, laboratory growth, and industry production (Rodrigues et al., 2010). Since the process causes discomfort, distress, and pain to animals, in vitro methods are preferred for producing mAbs. The volumetric productivity of mammalian cells cultivated in bioreactors has increased significantly over the past twenty years through improvements in media composition and process control (Wurm, 2004). Although there is still potential to increase the productivity of cells by controlling cell growth, the increase in amount has been insufficient (Kaufmann et al., 2003).
CD4 is a co-receptor that assists the T-cell receptor with an antigen-presenting cell to regulatory T cells, monocytes, macrophages, and dendritic cells. Anti-CD4 mAb has been used to treat many autoimmune disorders, including acute and relapsing experimental allergic encephalomyelitis and chronic eosinophilic lung inflammation (Doherty et al., 2009; Sriram et al., 1988). It has been used as an immunosuppressive agent for prolonging islet and pancreas allograft survival in mouse models (Mottram et al., 2000). Anti-CD4 mAb is also widely used in immunological and transplantation laboratories for studying autoimmune disorders; however, the antibody production in a hybridoma cell system is low, with a yield of only 12 mg/L cell culture, and thus cannot meet research and clinical needs.
Due to the complex nature of mAbs and their inherent heterogeneity, careful attention is required for product design and manufacturing to assure a safe, effective, and consistent mAb product. Consequently, mAb products are very expensive. With the rapidly growing demand for mAb-based products, new technologies are urgently needed to increase mAb production while reducing manufacturing costs.
Ultrasound is broadly defined as sound waves at a frequency above the normal hearing range, or a frequency greater than 20 kHz (Khanal et al., 2007). Ultrasound is traditionally used in medical diagnosis, such as fetal imaging which employs frequencies between 2 MHz and 18 MHz, and therapeutic treatment of injured muscles, ligaments and tendons, using frequencies between 1 to 5 MHz.
Ultrasonic stimulation creates “microcavitation” or the creation of minute bubbles in a liquid known as “microcavities.” With each sound wave, these bubbles expand and contract, creating tremendous force and turbulence on a microscopic scale. In some cases, this sound wave is powerful enough to collapse the cavities, which causes even more extreme turbulence, high temperatures, and free radicals in the vicinity of the former cavity. These collapses are powerful enough to dislodge or even destroy cells.
Ultrasonic applications rely on these processes. One common use of ultrasound is as an effective cleaning agent. If the intensity is high enough, collapse cavitation is the dominant factor in the cells' environment. This can strip or even kill harmful bacteria from a surface. The effectiveness of this technique has been proven by applying ultrasound to one end of a glass tube using frequencies around 100 kHz and intensities around 40 W/cm2. Approximately 88% of the bacteria were removed from the surface of the tube. Similar experiments have been carried out in a variety of situations, including stripping biofilms from reverse osmosis membranes. Ultrasound is now actively sold to laboratories as a cleaning aid.
As well as dislodging bacteria, very high intensity ultrasound (>10 W/cm2) has been used to kill suspended bacteria. This relies on collapse cavitation to rend the bacterial membrane.
Applications also exist for low intensity pulsed ultrasound (LIPUS) which generally utilizes an intensity of about 0.1-0.2 W/cm2. LIPUS has been used for repair of bone fractures, wound healing, and dental tissue regeneration (El-Bialy et al., 2007; Heckman et al., 1994; Rubin et al., 2001; Scheven et al., 2009); cell stimulation and differentiation (Yoon et al., 2009); stimulation of growth factors (Kobayashi et al., 2009); protein and fibroblast growth (Doan et al., 1999; Min et al., 2006; Sun et al., 2001; Wood et al., 1997; Zhou et al., 2004); dental tissue formation (Ang et al., 2010; Leung et al., 2004); stem cell proliferation (Gul et al., 2010); stem cell fate and lineage determination (Guilak et al., 2009); sonoporation including ultrasound-mediated gene delivery (Osawa et al., 2009); diagnostic applications (Harris, 2005; Ziskin, 1987); biomass pre-treatment before saccharification (Svetlana et al., 2010); and stimulation of bioactivity in a wide variety of cells, including human mesenchymal stem cells (Choi et al., 2011; Iwashina et al., 2006; Sun et al., 2001; Yun et al., 2009; Zhang et al., 2003; Zhou et al., 2004). It is believed that ultrasonic waves can improve the rate of bone growth and indeed, almost 80% of North American physiotherapists use ultrasonic emitters to promote recovery. However, only LIPUS is effective in this situation, with LIPUS devices being currently being marketed for this purpose (see for example, U.S. Pat. No. 4,530,360 to Duarte).
Use of low-intensity pulsed ultrasound to aid the healing of flesh wounds is described, for example, in U.S. Patent Application Publication No. 2006/0106424 A1 to Bachem. The method utilizes ultrasound to increase the phagocytotic action of the human body's macrophages. However, the method provides no solution for the use of ultrasound outside the confines of a wound.
U.S. Patent Application Publication No. 2003/0153077 A1 to Pitt et al. describes a method in which low-intensity ultrasound can stimulate the growth of biofilms and other cells. By balancing the beneficial turbulence produced by collapse cavitation with its accompanying negative effects, it was found that low-intensity ultrasound can improve growth rates of cells by up to 50%. The experimenters tested their findings on human and bacterial cells, using frequencies from about 20 kHz to about 1 MHz and intensities encompassing the range from 1 to 5000 mW/cm2. Unfortunately, though increased cell growth is beneficial to the fermentation process, the parameters investigated by this group do not provide the optimal rate of protein expression in fermentation processes.
Ultrasound promoted the growth of human skin fibroblasts by activating integrin receptors, RhoA/ROCK, and Src-ERK signaling cascades. In support of the proliferation-inducing effect of LIPUS, it has also been reported that LIPUS enhances the growth and production of proteoglycan in chondrocytes and intervertebral disc cells, possibly by enhancing growth factor-related genes (Iwashina et al., 2006; Kobayashi et al., 2009; Zhang et al., 2003). It was also recently shown that LIPUS supports the growth and colony-forming ability of the human umbilical cord- and bone marrow-derived (BM) mesenchymal stem cells (MSC) (Choi et al., 2011; Yun et al., 2009). LIPUS stimulation enhanced MSC yield and colony-forming ability at the early stage of primary cultures most likely through cell adhesion signals initiated from integrins, suggesting the possibility of employing LIPUS to obtain larger amounts of MSC for clinical applications. However, the application of LIPUS stimulation to expand hematopoietic stem/progenitor cells (HSPC) for clinical transplantation and cellular therapies has not yet been explored.